Product Specifications
Item# 1002: Recombinant tat HIV-1 IIIB
Concentration: See vial
Mass/vial: 100ug
Volume/vial: see vial
Diluent: 0.2 KCl, 5mM DTT, 50mM Tris, pH 8.0
Purity: >99%
Stabilizer: None
Preservative: None
Storage: -75°C
Physical State: Frozen Liquid
Stability: At least 12 months at -75°C
Applications: ELISA, Western ELISA, Anti-tat Drug Screening, Immunization Transcriptional Activation
Description: Full length (2 Exons, 86 amino acids) Recombinant HIV-1 IIIB tat produced in the E. coli Expression System.
Purification: This protein is purified by ion affinity and reverse phase HPLC to >99% purity, as determined by SDS-PAGE and HPLC.
Molecular weight: 12kD
Specificity: This protein binds to murine monoclonal antibodies of defined epitope specificity and human serum polyclonal antibodies in ELISA and Western ELISA.
Endotoxin: Less than 0.01 EU/mg of protein as determined by BioWhittaker Kinetic QCL Kit.
Biological Activity: The biological specificity of this protein was determined by LTR-CAT activation in scrapie-loaded HeLe Cells, and in U373 MG cells in the presence of 100μM chlorochine diphosphate. Tat concentrations in 1μg/ml range produced at least 25 fold increase in CAT activity. This protein inhibits DP4 activity in vitro and DP4-dependent T-cell activation in vivo.
Applications and Instructions for use
Recommended concentrations for use are approximate values. A dose dependent response assay should be performed to determine the optimal concentration for use in specific applications. Dilute tat stock solution in saline-citrate buffer (150mM NaCl, 50mM sodium citrate, pH 6.5) immediately before use. Tat readily oxidizes in buffer solutions which may change its LTR dependent transcriptional activation activity.
Transcriptional activation assays with tat are performed in 1-5μg/ml range. ELISA and Western ELISA require tat in 10-100ng protein range.
Related HIV-1 tat products: Item #1032 tat HIV-1 MN [click here to view], Item #1052 tat HIV-1 BAL and mutant bal [click here to view]
Biological Activity of Purified tat Protein
To examine whether purified tat retained biological activity, a modified scrape loading technique was used to introduce the protein into cell monolayers. In this procedure, either a monolayer culture of HeLa cells transfected 40 hr prior with plasmid pU3R-III or a monolayer culture of HeLa cell line that surface of the dish with a rubber policeman in the presence of the purified tat. Following the scraping procedure, cells were centrifuged, replated in fresh media, and CAT assays were performed 4 hr after addition of protein. As graphically elevated in cells that received that tat.
Glossary
Gene and Gene Products
Structural Proteins: Structural proteins – the products of gag, pol and env genes, which are essential components of the retroviral particle.
Regulatory Proteins: Regulatory proteins – tat and rev proteins of HIV/SIV and tax and rex proteins of HTLVs; essential for viral expression in infected cells.
Accessory Proteins: Accessory proteins – additional (non-regulatory) virion – and non virion-associated proteins produced by HIV/SIV retroviruses: vif, vpr, vpu, vpx, and nef. Although, the accessory proteins are not necessary for viral propagation in tissue culture, they have been conserved in the different isolates; this conservation and experimental observations suggest that their role in vivo is very important.
gag
gag – group-sepecifc antigens or capsid proteins; the precursor is the p55 myristoylated protein, which is processed to p17 (Matrix) p24 (Capsid) and p7 (NucleoCapsid) proteins by the viral protease. Other small proteins are generated from the gag polyprotein.
pol
pol – (p66) generates the viral enzymes protease (p11), reverse transcriptase (p51), endonuclease and integrase (p32) after the processing of a gag-pol precursor polyprotein by the viral protease; gag-pol precursor is produced by ribosome frameshifting.
env
env – viral glycoproteins produced as a precursor (gp160) and processed to the external glycoprotein (gp120) and the transmembrane glycoprotein (gp41). The mature proteins are held together by noncovalent interactions; as a result substantial amount of gp120 is released extracellularly. The external glycoprotein (gp120) contains the binding site for the CD4 receptor.
tat
tat – transactivator of HIV gene expression; one of the two necessary viral regulatory factors (tat and rev) for HIV gene expression. Two forms are known, tat-1 exon (minor form) of 72 amino acids, and tat-2 exon (major form) of 86 amino acids. The electrophoretic mobility of these two forms in SDS gels is anomalous; they are approximately 16 kD and 14 kD in weight. Low levels of both proteins are found in persistently infected cells. tat is localized primarily in the nucleolus/nucleus; it acts by binding to the TAR RNA element and activating transcription from the LTR promoter. Post-transcriptional effects of tat have been postulated.
rev
rev – the second necessary regulatory factor for HIV expression. A 19 kD phosphoprotein localized primarily in the nucleolus/nucleus, rev acts by binding to RRE and promoting the nuclear export, stabilization and utilization of the viral mRNAs containing RRE.
vif
vif – viral infectivity factor, typically 23 kD; required for the efficient transmission of cell-free virus in tissue culture. In the absence of vif, the produced viral particles are defective, while the cell-to-cell transmission of virus is not affected significantly. It has been reported that the cellular localization is in the Golgi (vif is not found in the virion).
nef
nef – approximately 27 kD non-virion protein found in the cytoplasm of infected cells. Potentially myristoylated and associated with the inner plasma membrane. One of the first HIV proteins to be produced in the infected cells, it is the most immunogenic of the accessory proteins and may be used in the future for diagnosis and staging of the disease. NEF is dispensable and probably suffers counter-selection during ex vivo viral propagation in vivo. Recent evidence suggests that SIV nef is required for viral propagation in vivo.
vpr
vpr – virion-associated protein of unknown function found in HIV-1, HIV-2, SIVmac, and SIVmnd; typically 15 kD. May be homologous to vpx. Also called “rap” for rapid.
vpu
vpu – protein that promotes extracellular release of viral particles. Found only in HIV-1. Integral membrane phosphoprotein of 16kd; similar to M2 protein of influenza virus. It may be involved in env maturation. It is not found in the virion.
vpx
vpx – virion protein of 12 kD found only in HIV-2 infection. (vpx may have some homology with vpr).
Related research paper:
Stimulation of HIV-1-neutralizing antibodies in simian HIV-IIIB-infected macaques
The humoral immune response to HIV type 1 (HIV-1) infection is inadequate in part because of the narrow range of virus-neutralizing antibodies elicited by the initially infecting virus and the failure to recognize subsequently arising virus variants.
Determination of Neutralizing Activity of Antibodies Against HIV-1 IIIB and MN.
A quantitative syncytium-forming microassay was employed for detection of the virus-neutralizing antibody response (13). Neutralization titers were determined by using two HIV-1 strains, HIV-1 IIIB and HIV-1 MN. A virus-syncytial-sensitive clone of CEM cells (CEM-SS) develops quantifiable, adherent syncytia (syncytium-forming units; SFUs) on a background of confluent, normal CEM monolayer in 4–6 days.
Statistical Analysis of Antibody Responses.
Viral antigen-binding antibodies and HIV-1 IIIB- and MN-neutralizing antibody titers were subjected to statistical analysis to determine the significance of changes observed after vaccination with mAb 1F7 or control mAb TEPC 183.
Analysis of Virus Neutralization Potency.
Blood samples from the three monkeys injected with mAb 1F7 and the control monkey injected with TEPC 183 were analyzed before, during, and after the vaccination regimen. Serial dilution of plasma specimens collected at different time points during the observation period were analyzed for virus-neutralizing antibody activity by using a quantitative syncytia-forming microassay (13) and HIV-1 strains IIIB and MN. The neutralization activity for HIV-1 IIIB and for HIV-1 MN was derived from five plasma dilutions at day 0 (prebleed), and four times after day 0 for each macaque.
Antibody neutralization activity for HIV-1 IIIB increased significantly after four inoculations of 1F7 in monkey 149-93 as determined at day 31
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DNA拓扑异构酶Ⅱ与抗肿瘤药物靶点的研究进展.doc(59.5k)
请规范发贴,求助贴请在标题前加【求助】,谢谢您的合作。——by草根
1970科家致癌RNA病毒发现种特殊RNA病毒聚合酶,该酶能RNA模板,根据碱基互补原则.程与般病毒转录向相反,故称逆转录,催化程酶称逆转录酶,发现哺乳物胚胎细胞裂淋巴细胞含.
逆转录病毒称携带逆转录酶病毒,先侵入宿主细胞病毒RNA模板,靠酶形DNA环化,合宿主细胞染色体原病毒形式宿主细胞代代传.
HIV(艾滋病病毒)典型逆转录病毒
伊立替康是喜树碱的半合成衍生物。喜树碱可特异性地与拓扑异构酶I结合,后者诱导可逆性单链断裂,从而使DNA双链结构解旋;伊立替康及其活性代谢物SN-38可与拓扑异构酶I-DNA复合物结合,从而阻止断裂单链的再连接。现有研究提示,伊立替康的细胞毒作用归因于DNA合成过程中,复制酶与拓扑异构酶I-DNA一伊立替康(或SN-38)三联复合物相互作用,从而引起DNA双链断裂。哺乳动物细胞不能有效地修复这种DNA双链断裂。
毒理研究
遗传毒性:伊立替康和SN-38在Ames试验中均未显示出致突变性。伊立替康在CHO细胞染色体畸变试验和小鼠微核试验中显示了致断裂作用。
生殖毒性:在啮齿动物多次给药试验中,可见雄性动物生殖器官萎缩。雌性大鼠静脉注射14C一伊立替康,其放射性可透过胎盘屏障,大鼠和家兔试验中,可见本品对胚胎和胎儿的毒性反应。大鼠静脉注射放射性标记的伊立替康后5分钟内,可在其乳汁中检测到放射性,给药4小时后乳汁中药物浓度可达到血药浓度的65倍;雌性大鼠在围产期静脉注射本品可引起仔鼠学习能力和雌鼠仔鼠体重的下降。
尚无足够的和严格控制的孕妇临床研究资料,若患者在孕期使用本品或在使用本品期间怀孕,应被告之对胎儿的潜在危害。有生育可能的妇女在本品给药期间应避免怀孕;母亲在接受本品治疗期间应停止哺乳。
致癌性:尚未进行伊立替康长期给药的致癌性研究,但进行了大鼠连续三周、每周一次静脉注射伊立替康2mg/kg和25mg/kg,然后恢复91周的试验(大鼠静脉注射伊立替康25mg/kg后,其Cmax和AUC分别约相当于人每周给药125mg/m2后的7倍和1.3倍),结果显示,子宫喇叭口处子宫内膜间质息肉和子宫内膜间质肉瘤发生率的增加有明显的剂量依赖性。
【药代动力学】
文献报导,人体静脉注射本品后,伊立替康的血浆浓度呈常指数消除。平均消除半衰期为6~12小时,活性代谢产物SN-3 8的消除半衰期为10~20小时。因为其内酯和羟基酸是化学平衡的,故活性内酯和SN-38的半衰期与完整的伊立替康和SN-38的半衰期相近。
在50~350mg/m2的剂量范围内,伊立替康吸收面积(AUC)与剂量呈线性递增关系:SN-38的AUC增加要小于剂量的增加。在90分钟内静脉滴注本品后1小时内,活性代谢产物SN-38达到最大浓度。伊立替康与血浆蛋白的结合率为 30%~68%,明显低于SN-38与血浆蛋白的结合率(大约95%)。伊立替康主要在肝内由羧酸酯酶转化为活性代谢产物SN-38,后者代谢为葡萄糖甙酸,活性为SN-38的1/50~1/100(由体内细胞毒性检测)。体内分布不明;药物及代谢产物经尿排泄:伊立替康为11%~20%,SN-38<1%,SN-38糖甙约3%。给药48小时后胆汁蓄积和经尿排泄的药25%~50%。
【贮 藏】
遮光,密闭保存。
【包 装】
西林瓶装,1瓶/盒,有40mg及100mg两种规格。
效 期】
24个月
【执行标准】
WS1-(X-166)-2005Z
【生产企业】
企业名称:辉瑞制药;安万特制药;齐鲁制药;江苏恒瑞医药股份有限公司向左转|向右转
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